Compound, agent and composition for the suppression of cancer growth

ABSTRACT

Novel  85 Rb-enriched rubidium salt compounds of general formula 1, below and novel compounds of general formula 2, below. Compositions that contain at least one of the novel compounds and optionally further contain an antitumor drug. Methods that entail administering such compounds and compositions to treat cancer, optionally in combination with a conventional form of cancer therapy, such as chemotherapy and radiation treatment. When administered with a conventional form of cancer therapy, the compounds and compositions of the invention may be administered before, simultaneously with, or after administration of the conventional form of cancer therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/680,584, filed on Jun. 5, 2018, and U.S. Provisional Application No.62/741,318, filed on Oct. 4, 2018, both of which are hereby incorporatedby reference herein in their entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of medicine and pharmacology,and more specifically, to novel methods, compounds and compositions forthe treatment of neoplastic diseases, melanoma in particular. Thecompound of the invention is a rubidium organic salt enriched for ⁸⁵Rbthat shows antitumor activity. The composition of the present inventioncomprises the ⁸⁵Rb-enriched salt optionally in combination withantitumor agents wherein the combination of components provides asynergistic suppression effect on cancer growth. The claimed compoundand composition based on the ⁸⁵Rb-enriched organic salt can be used forcancer treatment both individually and in combination with otherantitumor therapies. The invention can be used for the effectivetreatment of malignant diseases as it is characterized by high activityagainst cancer cells and has a reduced toxic effect on normal cells andthe whole organism in general.

BACKGROUND OF THE INVENTION

At present, cancer is the second leading cause of death globally, aftercardiovascular diseases. Statistics show that the incidence ofoncological diseases is growing every year, and today this problem isone of the greatest challenges for medical and pharmaceuticalindustries.

Methods of treating cancer include surgery, radiation therapy andchemotherapy. A surgical approach to the treatment of tumors is based onthe removal of the tumor burden and is attended by a high risk andstress associated with surgical intervention and postoperativecomplications. In addition, there is no guarantee that all of the canceris removed and that metastasis does not occur after removal of the tumortissue. Surgical removal of the primary tumor without stimulation of theimmune system, as a rule, leads to metastasis. Radiation therapy canlead to serious consequences for the patient's body, as it causesgenetic damage to normal healthy viable cells, has a carcinogenic effectand inhibits the patient's immune system.

Chemotherapy is another approach used to treat cancer. Chemotherapy is atype of cancer treatment that uses drugs to destroy cancer cells. Somechemotherapeutic drugs can be prescribed in a certain order depending onthe type of cancer. Although chemotherapy can be very effective in thetreatment of certain types of cancer, chemotherapeutic drugs canadversely affect not only tumor cells but also normal cells. Because ofthis, side effects are quite common with systemic chemotherapy. Acombined use of chemotherapeutic drugs can improve the suppression oftumor growth in a patient, but such combination chemotherapy does notreduce its adverse toxicity to the body. In addition, in many cases,chemotherapy causes severe side effects and carries the risk ofsuppression of the immune system.

Chemotherapy, in a significant number of cases, also produces severeside effects and carries a risk of suppression of the immune system. Inthis regard, there is currently a need for innovative methods oftreating cancer that would not produce such destructive effects on thepatient's body as described above.

An approach that involves the use of light isotopes for the treatment ofcancer in humans and animals is a topic for scientific research ofworldwide interest and is gaining ground every year. In particular, ithas been found out that natural water and most foodstuffs used by mancontain heavy isotopes of chemical elements. Each person, being acomplex biochemical system, fractionates heavy isotopes throughouthis/her life. As a result, accumulation of heavy isotopes in a humanbody starts from the moment of a person's birth. They gradually “embed”in the cells of the body which leads to a constant decrease in the rateof biological processes. One of the consequences of such accumulation isdiminishing of the body's ability to get rid of wastes, toxins and heavymetals which inevitably causes deterioration of health and generallyfeeling unwell, illnesses become more frequent, old age comes earlierand life shortens. In addition, heavy isotopes embed into DNA and RNAcells and disrupt the work of genetic apparatus of human cells which hasa harmful effect on the health of future generations.

A number of works demonstrate that the isotopic composition of tissuesand organs may serve as a diagnostic marker. In particular, the study ofratios of Cu and Zn isotopes in blood showed promisinginterrelationships with age, sex and pathologies. For example, anestimate of the ratio of Cu isotopes in serum is a new approach to thediagnosis and prognosis of the development of cirrhosis (M.Costas-Rodriguez et al., Isotopic analysis of Cu in blood serum bymulti-collector ICP-mass spectrometry: a new approach for the diagnosisand prognosis of liver cirrhosis, Metallomics 2015, 7. 491-498). Theisotopic composition of Zn in breast tissues makes it possible todiagnose cancer (F. Larner et al., Zinc isotopic compositions of breastcancer tissue, Metallomics 2015, 7. 107-112).

Patent application WO2007/140280 proposes an anticancer composition fortopical administration comprising cesium ions and/or rubidium ions aspharmaceutically acceptable salts that can be used for the treatment ofmalignant melanomas. The expediency of using this method is based on anapproach that involves changing the acidic pH of cancer cells to aslightly alkaline pH which puts the survival of a cancer cell at risk,and the formation of acidic and toxic materials, which usually occurs incancer cells, is neutralized and eliminated (Sartori, H E. Nutrients andcancer: an introduction to cesium therapy, Pharmacol. Biochem. Behav.1984; 21, Suppl. 1: 7-10). Thus mass-spectroscopic and isotopic studieshave shown that potassium, rubidium and cesium are absorbed by cancercells most efficiently. Glucose can still enter the cell, but oxygencannot. Thus the cell becomes anaerobic. In the absence of oxygen,glucose is fermented to lactic acid, and pH of the cell is reduced to 7and finally to 6.5. Cesium, rubidium and potassium, which create high pHvalues, are able to enter cells in this state and increase their pHvalue. Cesium and rubidium ions can change the ionic physiology of thecancer cell including inhibition of the transmembrane movement ofpotassium. It is assumed that cesium and rubidium effectively controlthe flow of potassium and bound hydrogen ion (H+) that affect allacid-dependent cancers and provide affinity to the site to selectivelyincrease the pH of the tumor microenvironment. Without wishing to bebound by theory, the inventors hypothesize that this provides selectivetumor modulation and jeopardizes the tumor's existence. The presentinvention provides, among other things, novel ⁸⁵Rb-enriched compounds, acomposition for oral administration, a composition for topicaladministration by itself as stand-alone treatment and provides acomposition for intravenous administration as a supplement to cancertherapy, all of which compositions contain the novel ⁸⁵Rb-enrichedcompounds, and methods that entail administering such compositions totreat cancer. In comparison with the state of the art (see, e.g., patentapplication WO2007/140280), the claimed invention is characterized byhigh efficiency and produces a systemic effect on the body.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide a compound ofFormula 1, diagrammed below.

A compound of Formula 1 is a rubidium salt wherein the rubidium isenriched for ⁸⁵Rb and wherein each of R₁ through R₁₄ is independentlyselected from H, OH, F, Cl, Br, I, C₁-C₆ alkyl, C₁-C₆ alkoxy, and NO₂.In one embodiment, R₁, R₂, R₄, R₅, R₆, R₈, R₁₀, Rn, and R₁₃ of Formula 1are all H and the remaining R groups are as defined above. In anotherembodiment, R₁, R₂, R₄, R₅, R₆, R₈, R₁₀, R₁₁, and R₁₃ of Formula 1 areall H, R₃ is selected from H, CH₃, OCH₃, and NO₂, R₇ and R₉ are eachindependently selected from H and OCH₃, and R₁₂ and R₁₄ are eachindependently selected from H, Br, I, and NO₂. In another embodiment,R₁, R₂, R₄, R₅, R₆, R₈, R₁₀, R₁₁ and R₁₃ of Formula 1 are all H, R₃ isselected from H, CH₃, OH, OCH₃ and NO₂, R₇ and R₉ are each independentlyselected from H and OCH₃, R₁₂ is selected from H, Br, I and NO₂, and R₁₄is selected from H, OH, Cl, Br, I and NO₂.

In certain specific embodiments, R₁, R₂, R₄, R₅, R₆, R₈, R₁₀, R₁₁, andR₁₃ of Formula 1 are all H, and

-   -   a) R₃ is CH₃ and R₇, R₉, R₁₂, and R₁₄ are all H (Compound 1),    -   b) R₃, R₇, R₉, R₁₂, and R₁₄ are all H (Compound 2),    -   c) R₃ is CH₃, R₁₄ is Cl, and R₇, R₉, and R₁₂ are all H (Compound        3),    -   d) R₃ is CH₃, R₁₄ is OH and R₇, R₉, and R₁₂ are all H (Compound        4),    -   e) R₁₄ is OH and R₃, R₇, R₉, and R₁₂ are all H (Compound 5),    -   f) R₃ is OH and R₇, R₉, R₁₂, and R₁₀ are all H (Compound 6),    -   g) R₁₄ is NO₂ and R₃, R₇, R₉, and R₁₂ are all H (Compound 7),    -   h) R₁₂ is Br, R₁₄ is NO₂ and R₃, R₇, and R₉ are all H (Compound        8),    -   i) R₃ and R₉ are both OCH₃, R₁₂ is Br, R₁₄ is NO₂ and R₇ is H        (Compound 9), or    -   j) R₃ and R₉ are both OCH₃, R₁₄ is NO₂ and R₇ and R₁₂ are both H        (Compound 10).

The term “⁸⁵Rb_(e)” is used herein to refer to rubidium that is enrichedfor ⁸⁵Rb, also referred to as “⁸⁵Rb-enriched rubidium.” Compound 1 aboveis also referred to herein as the ⁸⁵Rb-enriched rubidium organic salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine and as “E2+⁸⁵Rb_(e)”and “⁸⁵Rb_(e)-E2.” “E2” refers toN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine.

In any of the above compounds, the rubidium preferably is at least 75%⁸⁵Rb, more preferably at least 85% ⁸⁵Rb, and still more preferably atleast 95% ⁸⁵Rb, and in some embodiments is at least 99% ⁸⁵Rb, such as99.8% ⁸⁵Rb. “Rb that is N % ⁸⁵Rb” refers to Rb of which N % of the Rbatoms are the isotope ⁸⁵Rb.

It is another object of the invention to provide the compound of Formula2:

and salts thereof. Exemplary salts include the sodium salt, potassiumsalt, and rubidium salt.

In the compound of Formula 2, each of R₁ through R₁₄ is independentlyselected from H, OH, F, Cl, Br, I, C₁-C₆ alkyl, C₁-C₆ alkoxy, and NO₂.In one embodiment, R₁, R₂, R₄, R₅, R₆, R₈, R₁₀, R₁₁, and R₁₃ of Formula2 are all H and the remaining R groups are as defined above. In anotherembodiment, R₁, R₂, R₄, R₅, R₆, R₈, R₁₀, R₁₁, and R₁₃ of Formula 2 areall H, R₃ is selected from H, CH₃, OCH₃, and NO₂, R₇ and R₉ are eachindependently selected from H and OCH₃, and R₁₂ and R₁₄ are eachindependently selected from H, Br, I, and NO₂. In certain specificembodiments, R₁, R₂, R₄, R₅, R₆, R₈, R₁₀, R₁₁, and R₁₃ of Formula 2 areall H, and

-   -   a) R₃ is CH₃ and R₇, R₉, R₁₂, and R₁₄ are all H (Compound 11),    -   b) R₃, R₇, R₉, R₁₂, and R₁₄ are all H (Compound 12),    -   c) R₃ is CH₃, R₁₄ is Cl, and R₇, R₉, and R₁₂ are all H (Compound        13),    -   d) R₃ is CH₃, R₁₄ is OH and R₇, R₉, and R₁₂ are all H (Compound        14),    -   e) R₁₄ is OH and R₃, R₇, R₉, and R₁₂ are all H (Compound 15),    -   f) R₃ is OH and R₇, R₉, R₁₂, and R₁₄ are all H (Compound 16),    -   g) R₁₄ is NO₂ and R₃, R₇, R₉, and R₁₂ are all H (Compound 17),    -   h) R₁₂ is Br, R₁₄ is NO₂ and R₃, R₇, and R₉ are all H (Compound        18),    -   i) R₃ and R₉ are both OCH₃, R₁₂ is Br, R₁₄ is NO₂ and R₇ is H        (Compound 19), or    -   j) R₃ and R₉ are both OCH₃, R₁₄ is NO₂ and R₇ and R₁₂ are both H        (Compound 20).

Compound 11 above is also referred to herein asN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine and as “E2”.

In another aspect, the invention provides methods of synthesizing thecompounds of formula 1 and the compounds of formula 2, as diagrammedbelow.

Phase 1. Aryl sulfonation:

Phase 2. Acylation:

Phase 3. Obtaining rubidium complex. To prepare ⁸⁵Rb-enriched compounds,⁸⁵Rb_(e)Cl(⁸⁵Rb_(e) is 99% ⁸⁵Rb, for example) is used in the final stepshown below.

In another aspect, the invention provides a method of synthesizing acompound of formula 1 that uses a compound of formula 2 or a saltthereof as starting material or as an intermediate in the synthesis ofthe compound of formula 1. In an embodiment, the R groups of thecompound of formula 2 and salts thereof are the same as thecorresponding R groups of the compound of formula 1. In an embodiment,the synthetic method proceeds as outlined below:

This synthesis produces the potassium salt as an intermediate and therubidium salt as the product. In an analogous method, ⁸⁵Rb_(e)Cl can beused instead of RbCl to obtain a product in which the rubidium isenriched for ⁸⁵Rb.

In another aspect, the invention provides compositions that comprise oneor more compounds of general formula 1, above, or one or more compoundsof a sub-genus thereof as described above, or one or more of theabove-recited ⁸⁵Rb-enriched compounds 1-10. The invention also providescompositions that comprise a compound of general formula 2, above, or acompound of a sub-genus thereof as described above, or one or more ofthe above-recited compounds 11-20. The compositions include any typeknown in the art, including but not limited to liquid compositionsformulated for intravenous or other parenteral administration,compositions formulated for topical administration, and compositionsformulated for oral administration, such as, for example, tablets,pills, capsules, lozenges, granules. In certain embodiments, thecompositions of the invention comprise between 0.4 millimoles and 30millimoles of a compound of the invention, such as between 1 millimoleand 10 millimoles, or such as 1, 2, 5, 10, 20, 25, or 30 millimoles. Thecompositions of the invention further comprise one or more excipientsappropriate to the formulation. Intravenous formulations of theinvention comprise at least one of: an appropriate solvent, such aswater; a salt or ions such as sodium chloride, potassium chloride,potassium ion, sodium ion, chloride ion; a sugar such as glucose andsucrose; a buffer; other conventional excipients, such as DMSO. Topicalformulations of the invention include but are not limited to ointments,creams, lotions, salves and comprise at least one of: an appropriatevehicle; a penetration enhancer, such as DMSO and related analogues; andan emulsifier. Tablets of the invention comprise at least oneconventional excipient such as, for example: a filler (e.g. starches,lactose, sucrose, glucose); a binder (e.g. carboxymethylcellulose,gelatin, polyvinylpyrrolidone, sucrose); a disintegrating agent (e.g.calcium carbonate, alginic acid, sodium carbonate); a wetting agent(e.g. cetyl alcohol, and glycerol monostearate, sodium lauryl sulfate);a buffering agent; a lubricant (e.g. talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate); and acoating.

In another aspect, the invention provides a method of treating acondition that comprises administering a compound of the invention or acomposition of the invention, as set forth above. In an embodiment, thecondition treated is cancer, such as melanoma. In an embodiment, themethod of the invention is a method of treating cancer or suppressingcancer growth that comprises administering the ⁸⁵Rb-enriched rubidiumcompound of Formula 1 or composition that comprises it either alone orin combination with a conventional form of cancer therapy, such aschemotherapy (e.g. antitumor drugs, such as antitumor drugs currentlyknown and used in cancer therapy) or with other known cancer therapiessuch as radiation treatment. In various embodiments, the compound orcomposition of the invention is administered before, simultaneouslywith, or after the chemotherapy or other known anticancer therapy isadministered. When not administered simultaneously, the interval betweenadministrations is about 48 hours or less, such as 36 hours or less, or24 hours or less.

In another aspect, the invention provides a composition for use in thetreatment of a condition, in a human or in a veterinary animal, such asa dog, cat, cow or horse, wherein the composition is any of thecompositions described above. In an embodiment, the condition treated iscancer, such as melanoma. In various embodiments, the compositioncomprises one or more than one of the ⁸⁵Rb-enriched rubidium compoundsdescribed above, such as the ⁸⁵Rb-enriched rubidium compound ofFormula 1. In an embodiment, the composition further comprises anothersuitable active ingredient, such as an antitumor drug, such as anantitumor drug currently known and used in cancer therapy.

In another aspect, the invention provides a composition for use in thesuppression of cancer growth, wherein the composition is any of thecompositions described above. In an embodiment, the cancer growthsuppressed is melanoma. In various embodiments, the compositioncomprises one or more than one of the ⁸⁵Rb-enriched rubidium compoundsdescribed above, such as the ⁸⁵Rb-enriched rubidium compound ofFormula 1. In an embodiment, the composition further comprises anothersuitable active ingredient, such as an antitumor drug, such as anantitumor drug currently known and used in cancer therapy.

In another aspect, the invention provides a compound for use in thetreatment of a condition, in a human or in a veterinary animal, such asa dog, cat, cow or horse, wherein the compound is any of the⁸⁵Rb-enriched rubidium compounds of Formula 1 described above. In anembodiment, the condition treated is cancer, such as melanoma.

In another aspect, the invention provides a compound for use in thesuppression of cancer growth, wherein the compound is any of the⁸⁵Rb-enriched rubidium compounds of Formula 1 described above. In anembodiment, the cancer growth suppressed is melanoma.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the efficacy data (synergistic effect) in mouse B16melanoma cells (MM-4 cell line) for a composition that comprises the⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine and the antitumordrug dacarbazine (Example 2).

FIG. 2 shows the efficacy data in mouse melanoma cells (MM-4 cell line)for a composition that comprises the ⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine and the antitumordrug paclitaxel (antagonistic interaction was observed) (Example 2).

FIG. 3 shows the efficacy data (synergistic effect) in mouse melanomacells (MM-4 cell line) for a composition that comprises the⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine and the antitumordrug vinorelbine (Example 2).

FIG. 4 shows the efficacy data (synergistic effect) in mouse melanomacells (MM-4 cell line) for a method that comprises administering the⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine followed by theadministration (24 hours later) of an antitumor drug dacarbazine(Example 3).

FIG. 5 shows the efficacy data (synergistic effect) in mouse melanomacells (MM-4 cell line) for a method that comprises administering the⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine followed by theadministration (24 hours later) of paclitaxel (Example 3).

FIG. 6 shows the efficacy data (synergistic effect) in mouse melanomacells (MM-4 cell line) for a method that comprises administering the⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine followed by theadministration (24 hours later) of doxorubicin (Example 3).

FIG. 7 shows the efficacy data (synergistic effect) in mouse melanomacells (MM-4 cell line) for a method that comprises administering the⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine followed by theadministration (24 hours later) of vinorelbine (Example 3).

FIG. 8 shows the efficacy data (synergistic effect) for a method thatcomprises administering the ⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine followed by theadministration (24 hours later) of cisplatin (Example 3).

FIG. 9 shows the dynamics of Ehrlich ascites carcinoma (“EAC”) growth ina study on the in vivo antitumor activity of ⁸⁵Rb_(e)-E2 compound incombination with the chemotherapeutic agent Cisplatin (Example 5).

FIG. 10 shows the number of live cells in the ascites on the 15^(th) and22^(nd) day after inoculation of Ehrlich carcinoma cells (Example 5).

FIG. 11 shows the number of dead cells in the ascites on the 15^(th) and22^(nd) day after inoculation of Ehrlich carcinoma cells (Example 5).

FIG. 12 shows the volume of ascitic fluid on the 15^(th) and 22^(nd) dayafter inoculation of Ehrlich carcinoma cells (Example 5).

FIG. 13 shows the suppression of EAC tumor growth after it was exposedto the combined action of cisplatin and the experimental compoundcontaining stable isotope Rb⁸⁵ (Example 5).

FIG. 14 shows the number of animals in control and experimental groupswith tumor (Example 5).

FIG. 15 shows the number of animals in control and experimental groupswithout tumor (Example 5).

FIG. 16 shows the percentage of animals in control and experimentalgroups without tumor (Example 5).

FIG. 17 shows the survival rate of animals in control and experimentalgroups (Example 5).

FIG. 18 shows distribution of the combination index CI (the diagramshows 95% confidence interval CI).

(In the Figures, in numbers that contain commas, the comma indicates adecimal point—for example, “4,5” indicates “4.5” and “1,5” indicates“1.5”.)

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method, compound and composition forthe suppression, in humans and in veterinary animals, of cancer growth,melanoma in particular. The present invention makes it possible toeliminate shortcomings of the prior art technical solutions. Thecompounds of the invention include ⁸⁵Rb-enriched rubidium (“⁸⁵Rb_(e)”)salts (also referred to as a rubidium complex or compound) of generalFormula 1:

wherein each of R₁ through R₁₄ is independently selected from H, OH, F,Cl, Br, I, C₁-C₆ alkyl, C₁-C₆ alkoxy, and NO₂.

In an exemplary embodiment, Compound 1 of the invention, R₃ is CH₃ andthe remaining R groups are H. This compound is also referred to hereinas the ⁸⁵Rb-enriched rubidium organic salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine and as “⁸⁵Rb_(e)-E2”and “E2+⁸⁵Rb_(e).” “E2” refers toN-benzoyl-N-(4-toluenesulfonyl)-o-phenylenediamine. (In the Figures, theterms “E2+⁸⁵Rb”, “E2Rb85”, and “85RbE2” refer to ⁸⁵Rb_(e)-E2.) Compoundsof the present invention are prepared as disclosed herein and usingchemical synthetic methods disclosed in the literature.

Rubidium is a chemical element of the main subgroup of group 1, period5, of the periodic table, with atomic number 37. ⁸⁵Rb is a stableisotope which has a percent natural abundance of 72.2%. That is,naturally occurring rubidium consists of a mixture of rubidium isotopes.In a sample of naturally occurring rubidium, 72.2% of the rubidium atomsare isotope ⁸⁵Rb. As used herein, “⁸⁵Rb-enriched rubidium” is rubidiumthat consists of more than 72.2% ⁸⁵Rb. Thus, in compounds of the presentinvention, the rubidium is more than 72.2% ⁸⁵Rb, such as at least about75% (e.g. at least 75%), at least about 80% (e.g. at least 80%), atleast about 85% (e.g. at least 85%), at least about 90% (e.g. at least90%), or at least about 95% ⁸⁵Rb (e.g. at least 95% ⁸⁵Rb). In preferredembodiments of the compound, composition, and methods of the presentinvention, the rubidium is at least about 90% ⁸⁵Rb (e.g. at least 90%⁸⁵Rb) and may be over 99% ⁸⁵Rb, such as about 99.8% ⁸⁵Rb (e.g. 99.8%⁸⁵Rb). The compositions of the present invention comprise such⁸⁵Rb-enriched compounds, and the methods of the invention entailadministering such compositions. The term “about” as used hereinindicates plus or minus 3% of the subject amount (e.g., “about 80%”refers to the range from 77.6% to 82.4%).

A compound of Formula 1 is effective in suppressing cancer growth. Invitro, a compound of Formula 1 is used at a concentration of from about0.5 μg/ml to about 2 μg/ml of tissue culture medium and the antitumordrug is used at a dose of 1 ng/ml-40 μg/ml. The compound is administeredin vivo at a dose between about 0.05 mg of ⁸⁵Rb_(e)/kg and about 100 mgof ⁸⁵Rb_(e)/kg, preferably between about 1 mg of ⁸⁵Rb_(e)/kg and about20 mg of ⁸⁵Rb_(e)/kg, and more preferably between 1.25 and 12.5 mg⁸⁵Rb_(e) per kg body mass. In some embodiments, the ⁸⁵Rb_(e) compoundsof the invention are used in vivo at a dose of about 5 mg of⁸⁵Rb_(e)/kg, about 10 mg of ⁸⁵Rb_(e)/kg, or about 15 mg of ⁸⁵Rb_(e)/kg.In certain embodiments, these dosages are the amount administered daily.For example, where the ⁸⁵Rb_(e) compound is to be administered at a doseof 10 mg of ⁸⁵Rb_(e)/kg to a subject having a mass of 70 kg, theappropriate composition would comprise 700 mg of ⁸⁵Rb_(e) provided inthe form of the ⁸⁵Rb_(e) salt. Where the composition is a liquid to beinfused or injected, for example, the total amount of ⁸⁵Rb_(e) infusedor injected in the form of the ⁸⁵Rb_(e) salt would be 700 mg. Asindicated, the above doses are the dose of the ⁸⁵Rb_(e). The molarequivalent of the corresponding salt compound of the invention would begreater in mass. For a dosage range of between 1 mg of ⁸⁵Rb_(e)/kg and20 mg of ⁸⁵Rb_(e)/kg and a body weight range of 40 kg to 120 kg, acomposition of the invention contains a compound of Formula 1 in anamount equivalent to between 40 mg ⁸⁵Rb_(e) and 2400 mg ⁸⁵Rb_(e). For adosage range of between 1.25 mg of ⁸⁵Rb_(e)/kg and 12.5 mg of⁸⁵Rb_(e)/kg and a body weight range of 40 kg to 120 kg, a composition ofthe invention contains a compound of Formula 1 in an amount equivalentto between 50 mg ⁸⁵Rb_(e) and 1500 mg ⁸⁵Rb_(e). In certain embodiments,the dosage can be subdivided for administration more than once daily,and the composition can contain the corresponding fraction of the totaldaily dose, e.g. ½ the daily dose for twice daily administration, ⅓ thedaily dose for thrice daily administration.

A composition of the present invention comprises one or more compoundsof Formula 1, such as any of Compounds 1-10 (e.g. the ⁸⁵Rb-enrichedrubidium salt of N-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine),and optionally also contains one or more conventional antitumor drugs,for example, one or more antitumor drugs selected from the group thatincludes dacarbazine, doxorubicin, paclitaxel, vinorelbine andcisplatin, and also optionally contains one or more adjuvant agents. Inan embodiment of the invention, the composition contains a compound ofFormula I, such as Compound 1, and an antitumor drug selected fromdacarbazine and vinorelbine. In a composition of the invention thatcontains both the ⁸⁵Rb-enriched rubidium salt of formula I and theantitumor drug, the ⁸⁵Rb_(e) salt is present at the in vivo doses setforth above, and the antitumor drug is present at a dose that is between0.05 and 2.5 times the approved dosage (that is, at the dosage that theantitumor drug would have been prescribed if not administered inconjunction with a compound of the invention).

Compositions of the present invention otherwise are those conventionalforms known in the art for administration to humans or animals. Thecompositions of the present invention are prepared using methods withinthe general practice applied in the pharmaceutical industry, such as,for example, methods illustrated in the latest edition of Remington'sPharmaceutical Science Handbook, Mack Pub. N.Y., USA. Compositions ofthe invention comprise at least one pharmaceutically acceptable vehicleor excipient. These include, in particular, for example, diluents,fillers, disintegrants, solubilizing agents, dispersing agents,preservatives, wetting agents, preservatives, stabilizers, bufferingagents (e.g. phosphate, citrate, acetate, tartrate), suspending agents,emulsifiers, and penetration enhancing agents such as DMSO, asappropriate. Compositions of the invention preferably are solutions forinjection, such as intravenous injection. Water is preferably used as adosing vehicle and diluent in an injectable composition. Otherpharmaceutically acceptable solvents and diluents may also be used inaddition to or instead of water, such as saline, glycerol and ethanol. Acomplete description of pharmaceutically acceptable excipients can befound, for example, in Remington's Pharmaceutical Sciences (Mack Pub.,Co., N.J. 1991) or other standard pharmaceutical science texts, such asthe Handbook of Pharmaceutical Excipients (Shesky et al. eds., 8th ed.2017).

Large macromolecules that are slowly metabolized, such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, and copolymers of amino acids can also be used as vehicles forthe agent and composition.

In addition, compositions of the invention may be as described above,but comprise a compound of Formula 2, or a salt thereof, such as asodium, potassium, or rubidium salt (not enriched for ⁸⁵Rb) instead of,or in addition to, a compound of Formula 1.

The invention provides a method that comprises administering to a humanor to a veterinary animal a compound or composition of the invention andoptionally at least one conventional form of cancer therapy, such asanti-cancer radiation therapy or one or more antitumor drugs(abbreviated herein “AD”). The method may be used to treat cancer and/orsuppress cancer growth, such as melanoma. High efficiency of the methodis achieved when a compound of the invention, such as the ⁸⁵Rb-enrichedrubidium salt of N-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine(Compound 1), is administered in vivo at a dose as described abovefirst, and then, after an interval of time, an antitumor drug isadministered. In an embodiment, the antitumor drug is selected from thegroup that includes dacarbazine, doxorubicin, paclitaxel, vinorelbineand cisplatin. In preferred embodiments, the antitumor drug isadministered at its approved dosage (that is, at the dosage at which theantitumor drug would have been prescribed in the absence of a compoundof the invention). In other embodiments, the antitumor drug isadministered at some fraction of its approved dosage, such as one-tenth,one-fifth, one-fourth, one-third, or one-half the approved dosage. Forexample, where the antitumor drug is cisplatin, 1.5 mg/kg of cisplatinwould be administered three times at 48-hour intervals to provide theapproved dosage of cisplatin. The time between administration of the⁸⁵Rb_(e) salt and administration of the antitumor drug is preferablybetween about 12 and about 24 hours. In some embodiments, the intervalis between about 6 and about 36 hours, or between about 3 and about 48hours. Exemplary intervals include 12, 18, 24, 30, and 36 hours. Inalternative embodiments, a compound of the invention is administered atthe same time as an AD or after the AD at an interval as describedabove.

The antitumor drugs (ADs) referred to above include, but are not limitedto, nitrosoureas (e.g., carmustine (BCNU) and lomustine (CCNU)),alkylsulphonates (e.g., busulfan and treosulfan), triazenes (e.g.,dacarbazine, temozolomid), platinum-based compounds (e.g., cisplatin,carboplatin, oxaliplatin), vinca alkaloids (e.g., vincristine,vinblastine, vindesine and vinorelbine), taxoids (e.g., paclitaxel orpaclitaxel equivalents or paclitaxel analogs (such as docetaxel),anti-metabolites, DHFR inhibitors (e.g., methotrexatum,dichloromethotrexate, trimetrexate, edatrexate), and anthracyclines(e.g., daunorubicin, doxorubicin, pegylated liposomal doxorubicin,idarubicin, epirubicin, mitoxantrone). While an expected range of ADdosage is between about 1 ng/ml and 50 μg/ml, the AD drug's dosagetypically will be determined based on its known dosage when used withoutthe rubidium salt of the invention.

An advantage of the present invention is that the combination of the⁸⁵Rb-enriched rubidium salt of the invention and an antitumor drugprovide a synergistic effect. As a consequence, less of the antitumordrug can be used when used in combination with rubidium salt of theinvention than when the antitumor drug is used alone. The side effectsassociated with use of the antitumor drug therefore can be diminishedwhen used in combination with the rubidium salt of the invention.

An ⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine (Compound 1) wassynthesized by the present inventors. The enriched rubidium was 99.8%⁸⁵Rb. The inventors have surprisingly found that the said compound iseffective in suppressing cancer growth. The inventors of the presentinvention have developed and investigated the possibility andeffectiveness of using the said compound in combination with antitumordrugs when administered sequentially. They have further discovered thatan additive and even synergistic effect on the suppression of cancergrowth may be achieved by administering the ⁸⁵Rb-enriched rubidium saltof N-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine of formula Ifollowing the preliminary administration of one or more antitumor drugs(“ADs”).

For any compound, a therapeutically effective dose can be estimatedinitially from cell culture or animal model assays. Mice, rats, guineapigs, rabbits, dogs or pigs are commonly used as models in animaltesting. An animal model can be used to determine the appropriate rangeof concentrations and route of administration. Such information can thenbe used to determine appropriate doses and routes of administration inhumans. To estimate human equivalent dose, it is recommended to use thedosage conversion table given in the Guidance for Industry and theReviewers document (2002, U.S. Food and Drug Administration, Rockville,Md., USA). The exact effective dose to a patient will depend on variousconsiderations, including the severity of illness, the overall health ofthe patient, age, body weight and sex of the patient, nutrition, timeand frequency of administration, route of administration, combination(s)of medicaments, reaction sensitivity and tolerability/response totherapy. The exact dose can be determined by routine experiments andaccording to the attending physician's professional judgment anddiscretion.

The agent and ⁸⁵Rb_(e)-compound-containing composition of the presentinvention are preferably administered intravenously or intramuscularly.Other conventional routes of administration may also be used, includingother routes of injection and via oral and topical administration.

In an embodiment of the invention, one or more of dacarbazine,paclitaxel and vinorelbine are used as antitumor drugs included in thecomposition. The total dose of a compound of Formula 1 of the inventionis between about 1.25 mg ⁸⁵Rb_(e)/kg body weight and about 12.5 mg⁸⁵Rb_(e)/kg body weight, and the total dose of any of these threeantitumor drugs is between about 0.45 mg/kg body weight and about 4.5mg/kg body weight.

In an embodiment of the invention, the composition of the invention(preferably containing a compound of Formula 1, such as any of compounds1-10) is used to treat melanoma as a form of cancer. In anotherembodiment, a method of treating melanoma is provided that comprises thesequential administration of one or more antitumor drugs either beforeor after the administration of the composition of the invention. Theinterval between administration of one or more ADs and theadministration of the composition of the invention is between about 12hours and about 30 hours, preferably between about 20 hours and 28hours, more preferably about 24 hours, such as 24 hours.

Melanoma (or skin cancer) is a common type of cancer that occurs inpatients of any age. The disease typically starts in either thepigment-producing cells of the skin called melanocytes, which makemelanin, or from pigmented tissues, such as moles (also known aspigmented nevi). This form of cancer is characterized by rapiddevelopment. Along with squamous cell carcinoma and basal cellcarcinoma, melanoma is referred to as a malignant skin tumor. Mostfrequently it originates in the skin, less often in the retina of theeye and rarely in the mucous membranes of the oral cavity, genitalia orrectum. Melanoma is one of the most dangerous types of all humancancers, often recurring and spreading both hematogenously and throughlymphatic vessels to almost all organs in the body. A peculiar featureof this form of cancer is a weak immune response of an organism to thetumor, or even absence thereof, which is why very often melanomaprogresses rapidly. This type of pathology accounts for 10 percent ofall malignant skin lesions. It takes third place in frequency among allcancers in people belonging to the 15 to 39 age group.

The present inventors have surprisingly found that the compound ofFormula 1, such as the ⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N-(4-toluenesulfonyl)-o-phenylenediamine (Compound 1), incombination with an antitumor drug (AD), suppresses cancer growth. Inparticular, the compound of Formula 1, the agent based thereon and thecomposition increase survival in in vitro and in vivo studies in modelsystems.

An advantage of a compound of Formula 1 and a composition comprising itas described above is that their use enables reduction of toxic effectswhich are common to most methods based on the use of known cytostaticagents, while improving their efficacy in eradicating the establishedtumor. Due to the important physiological role of rubidium used in thecomposition and the method of producing the novel compound so that it isenriched for ⁸⁵Rb, in addition to antitumor effect, it is possible togain a number of additional advantages associated with the optimizationof the catalytic, structural and regulatory function of the organism.

High antitumor activity of a compound of Formula 1, in combination withan AD, in particular, high cytotoxic and cytostatic effects on melanomacells, was demonstrated in in vitro experiments on cell culture and invivo experiments in mice, wherein an additive and even synergisticeffect was achieved. Such synergy enables the use of lower doses ofchemotherapeutic agents thereby reducing their toxic effects on healthycells, as is common to most methods based on the use of known cytostaticagents, while improving their efficacy in eradicating the establishedtumor. Due to the important physiological role of rubidium used in thecomposition and the method of producing the novel compound, in additionto antitumor effect, it is possible to gain a number of additionaladvantages associated with the optimization of the catalytic, structuraland regulatory function of the organism.

It is especially important that the cytotoxic effect of AD on normalcells is reduced with a combined administration of AD and an ⁸⁵Rb_(e)salt compound of the invention. A relatively high antitumor activity ofthe methods of the invention, in particular, the ability to provide highcytotoxic and cytostatic effects on melanoma cells, was demonstrated inin vitro experiments on cell culture and in vivo experiments in mice.

The present invention is described more fully hereinafter by referenceto the following examples, which are presented by way of illustrationonly and should not be construed in any way to limit the scope of thepresent invention.

A synthetic scheme for preparing certain compounds of the invention andfor use according to the invention is set forth below.

Phase 1. Aryl sulfonation of o-phenylenediamine:

Phase 2. Acylation of N—R₃-phenylsulfonyl-o-phenylenediamine:

Phase 3. Obtaining of N—(R₁₂,R₁₄-benzoyl)-N′—(R₃-phenylsulfonyl)-o-phenylenediamine rubidium complex.To prepare ⁸⁵Rb-enriched compounds, ⁸⁵Rb_(e)C₁ (⁸⁵Rb_(e) is 99% ⁸⁵Rb,for example) is used in the final step shown below.

Compounds 1-10 were prepared by the above synthesis.

EXAMPLES Example 1. In Vitro Study of the Cytotoxic Activity of theCompound Comprising the ⁸⁵Rb-Enriched Rubidium Salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine of Formula IAccording to the Invention in B16 Melanoma Cells

In Vitro Effects of Experimental Compound ⁸⁵Rb_(e)-E2 on metabolicactivity of human Keratinocytes

(HaCaT line) E2 + Rb Concentration reference drug ⁸⁵Rb_(e) − E2 ofcompounds Metabolic activity of cells, % * 1 μg/ml 130 ± 2  0 0.5 μg/ml 103 ± 2.7 90 ± 1 0.25 μg/ml  95 ± 1.7  100 ± 8.7 0.13 μg/ml 87.5 ± 6.7 95.3 ± 10.7 62 ng/ml 97.3 ± 4.1 90.7 ± 2.7 31 ng/ml  99.2 ± 12.5 97.8 ±3.6 16 ng/ml 104.7 ± 3.2  98 ± 4 8 ng/ml —  114 ± 7.7In Vitro Assessment of the Effects of Experimental Compound ⁸⁵Rb_(e)-E2on metabolic Activity of Normal Human Fibroblasts (MTT Assay)

E2 + Rb Concentration reference drug ⁸⁵Rb_(e) − E2 of compoundsMetabolic activity of cells, % * 150 μg/ml 99.3 ± 3.2 72.8 ± 2.1 75μg/ml 97.7 ± 7.5 67.4 ± 4.7 38 μg/ml 98.8 ± 7.1 68.5 ± 1.8 20 μg/ml 96.8± 9.8  74 ± 1.3 10 μg/ml 104.6 ± 14.2 82.5 ± 1.3 5 μg/ml 103.4 ± 9.3 82.3 ± 3  2.5 μg/ml 128.2 ± 8.3  84.4 ± 4.6 1.25 μg/ml — 105.1 ± 2.3 In Vitro Assessment of Cytotoxic/Cytostatic Effects of ExperimentalCompound ⁸⁵Rb_(e)-E2 on Mouse Bone Marrow Cells

(MTT assay) E2 + Rb Concentration reference drug ⁸⁵Rb_(e) − E2 ofcompounds Metabolic activity of cells, % * 150 μg/ml 107.1 ± 3.8  0 75μg/ml 105.2 ± 7.8  18.5 ± 0.4 38 μg/ml 96.2 ± 4.4 19.1 ± 1.9 20 μg/ml 88 ± 3.5 24.2 ± 0.8 10 μg/ml 99.3 ± 4.7  45 ± 4.7 5 μg/ml 106.7 ± 11.890.2 ± 3.3 2.5 μg/ml 106.8 ± 6   97.4 ± 1.4 1.25 μg/ml 111.2 ± 6.6 119.1 ± 0.2 

The cytotoxic activity of the ⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine of formula I wasevaluated in the following experiment.

Materials and Methods: Mouse melanoma cells (MM-4 cell line) were seededinto 96-well plates (TPP, Italy) in DMEM (High Glucose w/L-Glutamine w/oSodium Pyruvate; Biowest, France) supplemented with 10% newborn calfserum (Biowest, France) and 40 mg/ml gentamicin (Sigma, USA) at aseeding density of 5×10⁴ cells/ml. The cells were then cultured in anincubator with a humidified atmosphere of 5% CO₂ in air at 37° C. 24hours after the cells were seeded, a compound according to the inventionthat comprises ⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine of formula I wasadded into the appropriate wells. ⁸⁵Rb_(e)-E2 was used at the followingdoses: 2.0 μg/ml, 1.0 μg/ml, 0.5 μg/ml (each is the final concentrationof ⁸⁵Rb_(e) after addition to the well). The cytotoxic effect wasdetermined by counting the number of live cells observed aftertreatment, expressed as a percentage of the number of live cellsobserved/counted in the control (cells seed in the well and nottreated). Control cells were not treated. For each concentration, theexperiment was carried out twice. The effects of the drugs wereevaluated visually, which allowed us to examine the morphologicalpattern of the cytotoxic action of the drugs, and by staining the cellswith crystal violet, followed by OD measurement at an excitationwavelength of 540 nm using a MultiScan spectrophotometer. Forcomparison, a parallel experiment was carried out with the use of aknown antitumor drug cisplatin and a compound of rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine of formula I whichcomprised a naturally-abundant mixture of isotopes of rubidium insteadof being enriched for the ⁸⁵Rb isotope. The results are presented inTable 1.

TABLE 1 Cytotoxic effect of ⁸⁵Rb-enriched and non-⁸⁵Rb-enriched rubidiumsalt of N-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine of formula I(cells used were Mouse melanoma cells (MM-4 cell line)) Number of cellsafter their treatment with the drug, % Dosage of ⁸⁵Rb_(e) as E2 +⁸⁵Rb_(e), μg/ml of ⁸⁵Rb_(e) 2.0  6.4 ± 0.3 1.0  8.3 ± 0.5 0.5 32.3 ± 0.7Dosage of Rb as E2 + Rb (naturally-abundant mixture of isotopes), μg/ml2.0 62.1 ± 0.8 1.0 84.9 ± 0.9 0.5 90.6 ± 0.9 Dosage of cisplatin, μg/ml10 μg/ml  2.6 ± 1.4 5 μg/ml 13.8 ± 1.2 2.5 μg/ml 24.6 ± 5.2 1.0 μg/ml51.7 ± 6.9 0.5 μg/ml  84 ± 3.3

As can be seen from the data presented, the efficacy of the agent of theinvention, which comprises the ⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine at a dose of 0.5-2.0μg rubidium/ml was comparable to cisplatin at a dose of 1.0-10 μg/ml inan in vitro model and exceeded the efficacy of the cisplatin used at adose of 0.5-5 μg/ml. The study has shown that the composition thatcontained the rubidium salt ofN-benzoyl-N-(4-toluenesulfonyl)-o-phenylenediamine of formula I, whichcomprises a naturally-abundant mixture of isotopes of rubidium insteadof being enriched for ⁸⁵Rb, and did not contain the ⁸⁵Rb-enrichedrubidium salt, had a significantly lower activity against tumor cells.

Example 2. In Vitro Study of the Cytotoxic/Cytostatic Activity of theComposition Comprising the ⁸⁵Rb-Enriched Rubidium Salt ofN-Benzoyl-N′-(4-Toluenesulfonyl)-o-Phenylenediamine Compound of FormulaI and Antitumor Drugs (AD) in B16 Melanoma Cells

In the in vitro experiment, the cytotoxic activity of the composition ofthe invention was studied in relation to the concentration of the⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N-(4-toluenesulfonyl)-o-phenylenediamine of formula I andantitumor drugs (“ADs”) with different mechanisms of action. Theexperiment allowed us to identify the phase-dependent effects of thecombined action of the drugs and to avoid undesirable combinations anddosing schedules thereof in the future. Accordingly, additional controlswere used in each case to objectively evaluate the results. Evaluationof the effects of the drugs was carried out visually, which allowed usto determine the morphological picture of the cytotoxic action of thedrugs, and by staining the cells for quantitative expression of theresults.

Materials and Methods: Mouse B16 melanoma cells (MM-4 cell line) wereseeded into 96-well plates (TPP, Italy), in DMEM (Biowest, France)supplemented with 10% newborn calf serum (Biowest, France) and 40 mg/mlgentamicin (Sigma, USA) at a seeding density of 5×10⁴ cells/ml. Thecells were then cultured in an incubator with a humidified atmosphere of5% CO₂ in air at 37° C. 24 hours after the cells were seeded, the saidcomposition, which contained various ratios and/or concentrations of thecomponents used, was added into the appropriate wells. The cells thenwere incubated at 37° C. and 5% CO₂ for an additional 72 hours (96 hoursin total after the cells were seeded). The following ADs were used inthe experiment:

-   -   1) Dacarbazine, Medac, GmbH, Germany    -   2) Paclitaxel, Actavis, Italy    -   3) Doxorubicin, Ebewe, Austria    -   4) Vinorelbine (Vinorelsin), Actavis, SindolPharma, Romania    -   5) Cisplatinum (also referred to as “cisplatin”), Ebewe, Austria

The results of the experiment were assessed visually, and the number ofcells was determined by staining the cells with crystal violet, followedby OD measurement at an excitation wavelength of 540 nm using aMultiSkan microplate spectrophotometer.

The effects of the complex action of various doses of the investigatedsubstances were evaluated using the concept of synergistic, additive andantagonistic effects of the drugs. The mathematics are detailed below.In short, an additive effect—the effect of the composition (E2+⁸⁵Rb_(e)and AD)—is equal to the sum of the effects of each separate substance. Asynergistic effect—the synergy of the two preparations (E2+⁸⁵Rb_(e) andAD)—is characterized by the fact that the sum of the effectssignificantly exceeds the sum of the effect of each individualsubstance. An antagonistic effect—the pure cytotoxic/cytostatic effectof the interaction of the two preparations (E2+⁸⁵Rb_(e) and AD) on tumorcells—is equal to zero (does not exceed the effect produced by eachsubstance individually).

The effects were calculated using the Chou-Talalay method according tothe following equation (“CI” refers to the “combination index”) (seeT.-C. Chou, Cancer Res. 70: 440-46 (Jan. 15, 2010):CI=(D)1/(Dx)1+(D)2/(Dx)2+(D)lx(D)2/(Dx)1/(Dx)2, where

(Dx)1 and (Dx)2 are the doses of preparations 1 and 2 that suppressgrowth of tumor cells by X % when used each individually; and

(D)1 and (D)2 are the doses of preparations 1 and 2 that suppress growthof tumor cells by X % when used in combination.

(As an example). The diagram (FIG. 18) shows the variation levels CI

Table (As an example). Scale of direction and forces of interactions ofbiologically active substances (physical factors) by Chou-Talalay RangeCI Description  <0.10 Very strong synergy 0.10-0.30 Strong synergies0.30-0.70 Synergism 0.70-0.85 Moderate synergies 0.85-0.90 Weak Synergy0.90-1.10 Additive effect 1.10-1.20 Weak antagonism 1.20-1.45 Moderateantagonism 1.45-3.30 Antagonism  3.30-10.00 Strong antagonism >10.00Very strong antagonism

A CI of 1 indicates that the preparations have an additive effect whenadministered together.

For example, in the above Table:

A CI<1 indicates a synergistic effect and CI>1 indicates an antagonisticeffect.

Tables 2-6 below shows the findings of the study.

Tables 2-6. Cytotoxic/cytostatic activity of the composition comprisingE2+⁸⁵Rb_(e) and AD (abbreviations explained below table).

Each table sets forth the % of MM-4 cells that survive in the presenceof the drug doses indicated in the in vitro experiments described above.The percentage is expressed as a percentage of the cells measured in thecontrol, which was not treated with either E2+⁸⁵Rb_(e) or AD (% ofsurviving MM-4 cells=100×MM-4 cells that survive in the presence of thedrug(s)/untreated MM-4 cells). Where both the ⁸⁵Rb-enriched rubidiumsalt (referred to as “E2+⁸⁵Rb_(e)”) and an AD were administered, theywere administered simultaneously. Table 2 data indicating synergy arepresented in chart form in FIG. 1; Table 3 data indicating synergy arepresented in chart form in FIG. 2; Table 5 data indicating synergy arepresented in chart form in FIG. 3. FIGS. 1-8 do not include the errorbars indicated in the text or Tables. For clarity, data indicatingadditive or antagonistic interactions between the AD and the⁸⁵Rb-enriched rubidium salt are omitted from the FIGS.

In the Tables, the following abbreviations have the following meanings:A: antagonistic effect; D: additive effect; S: synergistic effect.

TABLE 2 Results of experiments with dacarbazine and E2 + ⁸⁵Rb_(e) addedsimultaneously E2 + ⁸⁵Rb_(e) dose Dacarbazine dose (μg/ml) 0 40 μg/ml 20μg/ml 10 μg/ml 5 μg/ml 2.5 μg/ml 0 3.5 ± 0.7 7.8 ± 0.5 11.6 ± 3 18.1 ±0.2 21 ± 2.4 2 6.4 ± 0.3 0 (S) 0 (S) 0 (S) 0 (S) 1.2 ± 1.2 (S) 1 8.3 ±0.5 0 (S) 0 (S) 0 (S) 0 (S) 1 (S) 0.5 32.3 ± 0.7  1 (S) 1.3 ± 0.1 (S)2.6 ± 0.4 (S) 3.2 ± 0.2 (S) 4 ± 0.1 (S)

TABLE 3 Results of experiments with paclitaxel and E2 + ⁸⁵Rb_(e) addedsimultaneously E2 + ⁸⁵Rb_(e) dose Paclitaxel dose (μg/ml) 0 3 μg/ml 30ng/ml 15 ng/ml 7.5 ng/ml 3 ng/ml 0 1.9 ± 0.2 7 ± 0.8 16.1 ± 0.6 33.4 ±1.4 80 ± 2.3 2 6.4 ± 0.3 4.4 ± 0.2 (A) 8.6 ± 0.6 (A) 8.4 ± 0.6 (A) 8.3 ±0.1 (A) 5 ± 0.1 (S) 1 8.3 ± 0.5 2.3 ± 0.8 (A) 7.4 ± 0.1 (A) 7.8 ± 0.4(A) 7.2 ± 1.1 (A) 7.1 ± 0.1 (S) 0.5 32.3 ± 0.7  2.8 ± 0.2 (A) 10 ± 1.1(A) 10.4 ± 1.5 (A) 10.9 ± 0.2 (A) 22.7 ± 2.4 (A)

TABLE 4 Results of experiments with doxorubicin and E2 + ⁸⁵Rb_(e) addedsimultaneously E2 + ⁸⁵Rb_(e) dose Doxorubicin dose (μg/ml) 0 500 ng/ml300 ng/ml 200 ng/ml 175 ng/ml 150 ng/ml 0 0 3.1 ± 0.1 9.1 ± 1.1 13 ± 0.114.4 ± 1.1 2 6.4 ± 0.3 4.6 ± 1.2 (A) 6.2 ± 0.2 (A) 8.2 ± 1.1 (A) 8.7 ±0.6 (A) 7.9 ± 1.7 (A) 1 8.3 ± 0.5 3.8 ± 0.1 (A) 8.2 ± 1.4 (A) 16.6 ± 1.7(A) 15.2 ± 1.2 (A) 16.1 ± 0.1 (A) 0.5 32.3 ± 0.7  1.9 ± 0.2 (A) 6.9 ± 1(A) 13.6 ± 0.6 (A) 14.4 ± 0.6 (A) 14.6 ± 0.2 (A)

TABLE 5 Results of experiments with vinorelbine and E2 + ⁸⁵Rb_(e) addedsimultaneously E2 + ⁸⁵Rb_(e) dose Vinorelbine dose (μg/ml) 0 200 ng/ml50 ng/ml 25 ng/ml 5 ng/ml 1 ng/ml 0 1 2 ± 1 2 ± 0.8 9.5 ± 0.5 10 ± 0.1 26.4 ± 0.3 1.9 ± 0.4 (A) 1.5 ± 0.1 (A) 2.3 ± 0.5 (A) 2.2 ± 0.3 (S) 2 ±0.1 (S) 1 8.3 ± 0.5 0 (S) 0 (S) 0 (S) 1.9 ± 0.2 (S) 3.3 ± 0.3 (S) 0.532.3 ± 0.7  1 (A) 1 (S) 1 (S) 3.2 ± 0.1 (S) 4.9 ± 0.1 (S)

TABLE 6 Results of experiments with cisplatinum and E2 + ⁸⁵Rb_(e) addedsimultaneously E2 + ⁸⁵Rb_(e) dose Cisplatinum dose (μg/ml) 0 10 μg/ml 5μg/ml 2.5 μg/ml 1 μg/ml 0.5 μg/ml 0 2.6 ± 1.4 13.8 ± 1.2 24.6 ± 5.2 51.7± 6.9 84 ± 3.3 2 6.4 ± 0.3 1.9 ± 0.2 (A) 8.3 ± 1.3 (A) 8.1 ± 0.8 (A) 8.1± 1.3 (A) 8 ± 0.2 (A) 1 8.3 ± 0.5 1.2 ± 0.2 (A) 7.9 ± 0.5 (A) 7.8 ± 0.7(A) 7.9 ± 0.1 (A) 8 ± 0.1 (A) 0.5 32.3 ± 0.7  2.2 ± 0.2 (A) 11.8 ± 0.7(A) 15.3 ± 3.2 (A) 22.1 ± 0.4 (A) 22.3 ± 0.3 (D)

As can be seen from Tables 2-6 above, a synergistic effect was observedin the composition that comprised E2+⁸⁵Rb_(e) at a dose of 0.5-2.0 μg/mland dacarbazine at a dose of 2.5 to 40 μg/ml, a synergistic effect ofE2+⁸⁵Rb_(e) and paclitaxel composition was observed with E2+⁸⁵Rb_(e) at1.0 μg/ml and 2.0 μg/ml and paclitaxel at 3 ng/ml and the compositioncomprising E2+⁸⁵Rb_(e) and doxorubicin showed no synergistic effect. Asfor E2+⁸⁵Rb_(e) and vinorelbine composition, the synergistic effect wasobserved with E2+⁸⁵Rb_(e) at 0.5-1.0 μg/ml and vinorelbine at 1-200ng/ml. The composition of E2+⁸⁵Rb_(e) and cisplatin had an additiveeffect only in combination of E2+⁸⁵Rb_(e) and cisplatin both used at adose of 0.5 μg/ml.

The above results indicate that the composition comprising the⁸⁵Rb-enriched salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine of formula I at adose of 0.5-2.0 μg/ml and an antitumor drug selected from the groupconsisting of dacarbazine (triazene agent) and vinorelbine (vincaalkaloid) at a dose of 1 ng/ml to 40 μg/ml exhibits strong synergismwith respect to cytotoxic activity against tumor cells.

Example 3. In Vitro Study of the Effectiveness of the Claimed Method inB16 Melanoma Cells: Measuring the Effect of Two Drugs AdministeredSequentially 24 Hours Apart (⁸⁵Rb-Enriched Rubidium Salt First, ADSecond (Scheme 1 Below), and AD First, ⁸⁵Rb-Enriched Rubidium SaltSecond (Scheme 2 Below))

The effectiveness of the method of the invention that comprisesadministering antitumor drugs and ⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N-(4-toluenesulfonyl)-o-phenylenediamine of formula I wasmeasured in the experiment. A study similar to that described in Example2 was carried out, except that the ⁸⁵Rb-enriched rubidium salt and theAD were administered 24 hours apart. Both orders were tested: ADadministered first (Scheme 2, below), and AD administered second (Scheme1, below).

Materials and Methods: Mouse melanoma cells (MM-4 cell line) were seededinto 96-well plates (TPP, Italy), in DMEM (Biowest, France) supplementedwith 10% newborn calf serum (Biowest, France) and 40 mg/ml gentamicin(Sigma, USA) at a seeding density of 5×10⁴ cells/ml. The cells were thencultured in an incubator with a humidified atmosphere of 5% CO₂ in airat 37° C. The experiment was carried out in accordance with the threeschemes given below. The experiment was started 24 hours after the cellswere seeded. For this purpose, the agents used in the method wereintroduced into the appropriate wells. The following schemes ofintroduction were used for the study:

Scheme 1) The ⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine of formula I wasintroduced at the first stage in the following amounts: 2.0 μg/ml, 1.0μg/ml, 0, 5 μg/ml, and one antitumor drug was introduced at the secondstage (after 24 hours). The following ADs were used in the experiment:

-   -   Dacarbazine, Medac, GmbH, Germany (doses used: 2.5, 5, 10, 20,        40 μg/ml)    -   Paclitaxel, Actavis, Italy (doses used: 3.0, 7.5, 15, 30 ng/ml,        30 μg/ml)    -   Doxorubicin Ebewe, Austria (doses used: 150, 175, 200, 300, 500        ng/ml    -   Vinorelbine (Vinorelsin), Actavis, SindolPharma, Romania (doses        used: 1, 5, 25, 50, 200 ng/ml)    -   Cisplatinum Ebewe, Austria (doses used: 0.5, 1.0, 2.5, 5.0, 10        μg/ml)

Scheme 2) An AD was introduced at the first stage at doses as indicatedin Scheme 1 above and ⁸⁵Rb-enriched rubidium salt ofN-benzoyl-N′-(4-toluenesulfonyl)-o-phenylenediamine of formula I wasintroduced at the second stage (after 24 hours) at the following doses:2.0 μg/ml, 1.0 μg/ml, 0.5 μg/ml.

After all the agents were added into the wells, the cells were incubatedat 37° C. and 5% CO₂ for another 72 hours (96 hours in total after thecells were seeded).

As in example 2, the cytotoxic effect was determined as the percentageof the cell number with respect to the control after their treatment inaccordance with the claimed method. The effects of the drugs wereevaluated visually, which allowed us to examine the morphologicalpattern of the cytotoxic action of the drugs, and by staining the cellswith crystal violet, followed by OD measurement at an excitationwavelength of 540 nm using a MultiSkan microplate spectrophotometer.

The effects of the complex action of various doses of the investigatedsubstances were evaluated using the concept of synergistic, additive andantagonistic effects of the drugs, as for Example 2, and calculating CIas described in Example 2.

Tables 7-16 below show the findings of the study. Each table sets forththe % of MM-4 cells that survived in the presence of the drug dosesindicated in the in vitro experiments described above, expressed as apercentage of the cells in the control wells.

The data from table 7 is displayed in graph form in FIG. 4.

The data from table 8 is displayed in graph form in FIG. 5.

The data from table 9 is displayed in graph form in FIG. 6.

The data from table 10 is displayed in graph form in FIG. 7.

The data from table 11 is displayed in graph form in FIG. 8.

The FIGS. do not include the error bars indicated in the text or Tables.For clarity, data indicating additive or antagonistic interactionsbetween the AD and the ⁸⁵Rb-enriched rubidium salt are omitted from theFIGS.

In the Tables, the following abbreviations have the following meanings:A: antagonistic effect; D: additive effect; S: synergistic effect.

Tables 7-11. Effectiveness of the method comprising administeringE2+⁸⁵Rb_(e) first and AD second, 24 hours after E2+⁸⁵Rb_(e).(Abbreviations explained below table). In Tables 7-11, the E2+⁸⁵Rb_(e)dose indicated is the dose of ⁸⁵Rb_(e) in the form of the ⁸⁵Rb_(e)-E2salt.

TABLE 7 Results of experiments with dacarbazine added 24 hours afterE2 + ⁸⁵Rb_(e) E2 + ⁸⁵Rb_(e) dose dacarbazine dose (μg/ml) 0 40 μg/ml 20μg/ml 10 μg/ml 5 μg/ml 2.5 μg/ml 0 8.4 ± 1.2 12.9 ± 2.9 19 ± 4 30 ± 2.440.7 ± 0.7 2 6.4 ± 0.3 0 (S) 1.3 ± 0.3 (S) 1.6 ± 0.3 (S) 1.9 ± 0.6 (S) 2± 0.7 (S) 1 8.3 ± 0.5 0 (S) 1.1 ± 0.1 (S) 1.5 ± 0.4 (S) 1.9 ± 0.1 (S)2.5 ± 0.2 (S) 0.5 32.3 ± 0.7  4.9 ± 0.1 (S) 5.5 ± 1.1 (S) 5.9 ± 0.1 (S)7.3 ± 0.1 (S) 9.9 ± 0.9 (S)

TABLE 8 Results of experiments with paclitaxel added 24 hours after E2 +⁸⁵Rb_(e) E2 + ⁸⁵Rb_(e) dose paclitaxel dose (μg/ml) 0 3 μg/ml 30 ng/ml15 ng/ml 7.5 ng/ml 3 ng/ml 0 15.3 ± 1.6 29.2 ± 4.4 37 ± 4.5 49 ± 1 87.7± 6.7 2 6.4 ± 0.3 2.6 ± 0.2 (S) 5 ± 0.2 (S) 5.5 ± 0.4 (S) 5.4 ± 0.2 (S)5.4 ± 0.3 (S) 1 8.3 ± 0.5 2.2 ± 0.6 (S) 6.4 ± 1 (S) 7 ± 0.5 (S) 4.8 ±0.2 (S) 5.4 ± 0.4 (S) 0.5 32.3 ± 0.7  5.5 ± 0.1 (S) 9.1 ± 1.3 (S) 10.1 ±0.2 (S) 11 ± 1.1 (S) 19.3 ± 0.5 (S)

TABLE 9 Results of experiments with doxorubicin added 24 hours afterE2 + ⁸⁵Rb_(e) E2 + ⁸⁵Rb_(e) dose doxorubicin dose (μg/ml) 0 500 ng/ml300 ng/ml 200 ng/ml 175 ng/ml 150 ng/ml 0 5.3 ± 0.9 12.2 ± 0.5 24.8 ±5.5 27.9 ± 6.9 35.5 ± 0.8 2 6.4 ± 0.3 16 (A) 4 ± 1.6 (A) 4.6 ± 0.7 (D)7.1 ± 1.8 (A) 7.4 ± 1.3 (A) 1 8.3 ± 0.5 1.1 ± 0.1 (S) 3.6 ± 0.1 (S) 5.3± 0.5 (S) 5.6 ± 0.3 (S) 5.9 ± 0.3 (S) 0.5 32.3 ± 0.7  5.2 ± 0.6 (A) 11.3± 0.1 (A) 12.7 ± 0.8 (A) 16 ± 0.9 (A) 19 ± 2.7 (A)

TABLE 10 Results of experiments with vinorelbine added 24 hours afterE2 + ⁸⁵Rb_(e) E2 + ⁸⁵Rb_(e) dose vinorelbine dose (μg/ml) 0 200 ng/ml 50ng/ml 25 ng/ml 5 ng/ml 1 ng/ml 0 5.6 ± 0.6 6.4 ± 0.4 7.9 ± 0.5 17.9 ±0.9 26 ± 2.5 2 6.4 ± 0.3 0 (S) 1.2 ± 0.1 (S) 2.4 ± 0.6 (S) 2.4 ± 0.6 (S)2 ± 0.1 (S) 1 8.3 ± 0.5 1.1 ± 0.1 (S) 1.7 ± 0.7 (S) 1.9 ± 0.9 (S) 2.4 ±0.1 (S) 2 ± 0.5 (S) 0.5 32.3 ± 0.7  4.5 ± 0.2 (S) 6.1 ± 1.1 (A) 4.3 ±0.3 (S) 8.2 ± 0.7 (S) 8 ± 0.4 (S)

TABLE 11 Results of experiments with cisplatinum added 24 hours afterE2 + ⁸⁵Rb_(e) E2 + ⁸⁵Rb_(e) dose cisplatinum dose (μg/ml) 0 10 μg/ml 5μg/ml 2.5 μg/ml 1 μg/ml 0.5 μg/ml 0 19.4 ± 0.9 35.2 ± 3.3 34 ± 2.6 70 ±3.6 84.6 ± 3.5 2 (μg/ml) 6.4 ± 0.3 3 ± 0.1 (D) 5.9 ± 0.5 (A) 5 ± 0.2 (A)4.8 ± 1 (S) 3.7 ± 0.7 (S) 1 (μg/ml) 8.3 ± 0.5 1.9 ± 0.2 (S) 3.5 ± 1.2(S) 3.5 ± 0.1 (S) 4.4 ± 0.7 (S) 4.9 ± 0.4 (S) 0.5 (μg/ml) 32.3 ± 0.7 7.1 ± 0.9 (A) 15.8 ± 0.1 (A) 16.9 ± 1 (A) 20.9 ± 0.6 (S) 20 ± 1.5 (S)A—antagonistic effect, D—additive effect, S—synergistic effect

Tables 12-16. Effectiveness of the method comprising administering ADfirst and E2+⁸⁵Rb_(e) second, 24 hours after AD. In Tables 12-16, theE2+⁸⁵Rb_(e) dose indicated is the dose of ⁸⁵Rb_(e) in the form of the⁸⁵Rb_(e)-E2 salt.

TABLE 12 Results of experiments with E2 + ⁸⁵Rb_(e) added 24 hours afterdacarbazine E2 + ⁸⁵Rb_(e) dose dacarbazine dose (μg/ml) 0 40 μg/ml 20μg/ml 10 μg/ml 5 μg/ml 2.5 μg/ml 0 3.5 ± 0.7 7.8 ± 0.5 11.6 ± 3 18.1 ±0.2 21 ± 2.4 2 17.7 ± 0.8 0 (S) 2 ± 0.2 (S) 1.8 ± 0.1 (S) 2 ± 0.1 (S)2.9 ± 0.1 (S) 1  19 ± 0.5 1 ± 0.1 (S) 2 ± 0.1 (S) 2 ± 0.1 (S) 2 ± 0.1(S) 2 ± 0.1 (S) 0.5 51 ± 1 3.5 ± 0.3 (A) 3.9 ± 0.5 (S) 4.1 ± 0.6 (S) 5.6± 1.1 (S) 9.3 ± 1 (S)

TABLE 13 Results of experiments with E2 + ⁸⁵Rb_(e) added 24 hours afterpaclitaxel E2 + ⁸⁵Rb_(e) dose paclitaxel dose (μg/ml) 0 3 μg/ml 30 ng/ml15 ng/ml 7.5 ng/ml 3 ng/ml 0 1.9 ± 0.2 7 ± 0.8 16.1 ± 0.6 33.4 ± 1.4 80± 2.3 2 17.7 ± 0.8 5.5 ± 1.5 (A) 10.2 ± 1 (A) 12.4 ± 3.8 (A) 13.3 ± 1.4(A) 23.8 ± 2.4 (A) 1  19 ± 0.5 3.3 ± 0.6 (A) 7 ± 0.9 (A) 7.5 ± 0.9 (S)7.9 ± 0.5 (S) 11.2 ± 0.9 (S) 0.5 51 ± 1 4.1 ± 0.3 (A) 10.7 ± 2.7 (A)13.9 ± 1.8 (D) 13.3 ± 1.7 (S) 37 ± 3 (A)

TABLE 14 Results of experiments with E2 + ⁸⁵Rb_(e) added 24 hours afterdoxorubicin E2 + ⁸⁵Rb_(e) dose doxorubicin dose (μg/ml) 0 500 ng/ml 300ng/ml 200 ng/ml 175 ng/ml 150 ng/ml 0 0 3.1 ± 0.1 9.1 ± 1.1 13 ± 0.114.4 ± 1.1 2 17.7 ± 0.8 4.7 ± 1.3 (A) 9.7 ± 1.9 (A) 17.3 ± 0.3 (A) 17.9± 2.7 (A) 14 ± 0.8 (A) 1  19 ± 0.5 1.8 ± 0.8 (A) 4.4 ± 0.1 (A) 9.8 ± 1.9(A) 12.7 ± 2.6 (A) 12.5 ± 2.7 (A) 0.5 51 ± 1 0 (A) 5.3 ± 0.8 (A) 13.7 ±0.3 (A) 13.7 ± 1.1 (A) 13.1 ± 0.8 (A)

TABLE 15 Results of experiments with E2 + ⁸⁵Rb_(e) added 24 hours aftervinorelbine E2 + ⁸⁵Rb_(e) dose vinorelbine dose (μg/ml) 0 200 ng/ml 50ng/ml 25 ng/ml 5 ng/ml 1 ng/ml 0 1 2 ± 1 2 ± 0.8 9.5 ± 0.5 10 ± 0.1 217.7 ± 0.8 0 (S) 0 (S) 1.5 ± 0.5 (S) 3 ± 0.4 (S) 3 ± 0.5 (S) 1  19 ± 0.50 (S) 0 (S) 1 (S) 2.7 ± 0.4 (S) 2.8 ± 0.1 (S) 0.5 51 ± 1 0 (S) 0 (S) 1(S) 4 ± 1.5 (S) 8.1 ± 1.5 (S)

TABLE 16 Results of experiments with E2 + ⁸⁵Rb_(e) added 24 hours aftercisplatinum E2 + ⁸⁵Rb_(e) dose cisplatinum dose (μg/ml) 0 10 μg/ml 5μg/ml 2.5 μg/ml 1 μg/ml 0.5 μg/ml 0 2.6 ± 1.4 13.8 ± 1.2 24.6 ± 5.2 51.7± 6.9 84 ± 3.3 2 17.7 ± 0.8 1.9 ± 0.9 (A) 10.1 ± 0.1 (A) 16.4 ± 1.1 (A)13.6 ± 1 (S) 11.4 ± 0.3 (S) 1  19 ± 0.5 0 (S) 11 ± 1.7 (D) 12.1 ± 1.2(S) 12.5 ± 1 (S) 13.2 ± 1.5 (S) 0.5 51 ± 1 0 (S) 7.4 ± 0.3 (S) 15 ± 1.7(S) 25.4 ± 2.8 (A) 25.1 ± 2.7 (S) A—antagonistic effect, D—additiveeffect, S—synergistic effect

As can be seen from the data, the most effective administration regimenstested entailed the separate administration of E2+⁸⁵Rb_(e) and AD. Themost pronounced cytotoxic effect was in the case of delayedadministration of E2+⁸⁵Rb_(e) and AD. In particular, the synergisticeffect was observed in many combinations used in the experiment where ADwas administered 24 hours after E2+⁸⁵Rb_(e). When E2+⁸⁵Rb_(e) wasadministered 24 hours after AD, a synergistic effect in E2+⁸⁵Rb_(e) andpaclitaxel and E2+⁸⁵Rb_(e) and cisplatin combinations was observed onlywith the use of a few specific doses, while combinations withdoxorubicin showed only antagonistic effect. The method that entailsadministering the AD 24 hours after E2+⁸⁵Rb_(e) is preferred for thedrugs tested, although, as mentioned, the reverse order yielded synergyin certain dosage combinations. Another interesting property which wasalso demonstrated was that some chemotherapeutic drugs produced the besteffect at lower concentrations of the compound used, which makes itpossible to reduce the amount of an AD to be administered withoutreducing its efficacy.

Example 4: Reduced Toxicity of Compounds of the Invention

In Vitro Assessment of the Effects of E2+⁸⁵Rb_(e) on Metabolic Activityof Normal Human Fibroblasts (MTT Assay)

Concentration E2 + Rb of compounds reference drug E2 + ⁸⁵Rb_(e) of E2series Metabolic activity of cells, % * 150 μg/ml 99.3 ± 3.2 72.8 ± 2.175 μg/ml 97.7 ± 7.5 67.4 ± 4.7 38 μg/ml 98.8 ± 7.1 68.5 ± 1.8 20 μg/ml96.8 ± 9.8  74 ± 1.3 10 μg/ml 104.6 ± 14.2 82.5 ± 1.3 5 μg/ml 103.4 ±9.3  82.3 ± 3  2.5 μg/ml 128.2 ± 8.3  84.4 ± 4.6 1.25 μg/ml — 105.1 ±2.3  * in comparison with the control group - 100% of living cells

Proliferative and metabolic activity of cells was determined by thecolorimetric method.

The metabolic activity of the cells (i.e., the number of living cells)was assessed by MTT staining.

24 hours after the last reagent, the cells were seeded at aconcentration of 1×10⁴ cells/well in wells of a 96-well plate incomplete DMEM nutrient medium with 10% FBS and 40 μg/ml gentamicin. Thecells were cultured in a humidified atmosphere at 5% CO₂ and 37° C. for24 hours. After 24 hours, various doses of experimental substances wereadded to the respective wells. The cells were incubated at 37° C. and 5%CO₂ for an additional 72 hours.

Upon completion of the incubation with the preparations, theproliferative and metabolic activity of the cells in the experiment wasevaluated by the colorimetric method by staining MTT cells:

10 μl of the MTT solution (5 mg/ml of the dye in phosphate-bufferedsaline) was added to each well of the plate; the plate was incubated ina CO₂ incubator at 37° C. for 3 hours. After that, the medium wasremoved from the wells, and the formed tetraformazan crystals weredissolved in 100 μl of dimethylsulfoxide.

When MTT was used, the results were evaluated using a multi-wellspectrophotometer at an excitation wavelength of 540 nm. The percentageof viable cells was calculated by the formula:IR=(A540(experiment)/A540(control))×100%.

Thus, the method for the suppression of cancer growth according to theinvention can be highly effective in eradicating tumor cells. Theobserved synergistic effect of the drug interaction makes it possible tocontrol tumor cells using lower concentrations of chemotherapeuticagents than when such agents are used alone.

Example 5: Study on the In Vivo Antitumor Activity of ⁸⁵Rb_(e)-E2Compound in Combination with a Chemotherapeutic Agent (Cisplatin)Against Mouse Model of Breast Cancer (Ehrlich Ascites Carcinoma)

Brief Description of the Study

Outbred mice were administered Ehrlich ascites carcinoma cells (EAC)intraperitoneally (IP) at a dose of 2.5×10⁵ cells in 0.25 ml of normalsaline solution. The compounds under study, 1)⁸⁵Rb_(e)E2, was dissolvedin deuterium-depleted water and 2) Cisplatin (Teva, the Netherlands),was dissolved in normal saline solution. The drugs were administeredstarting from the first day after mice were inoculated with tumor cells,using a microinjection syringe, at the following doses: 1)⁸⁵Rb_(e)-E2 ata dose of 750 μg/mouse (weighing 20 g) in a volume of 0.3 ml as 3 IPinjections (1 injection—250 μg/mouse) on the 1st, 4^(th) and 7^(th) dayof the experiment; 2) Cisplatin at a dose of 4.5 mg/kg or 1.5 mg/kg as 3IP injections on the 2^(nd), 5^(th) and 8^(th) day of the experiment.The dynamics of tumor growth in the experimental animals was observed(based on the volume of ascites in the peritoneal cavity) after thetumor cell inoculation and for 31 days of the experiment. On the 15^(th)and 22^(nd) day after IP administration of tumor cells, all the fluidaccumulated in each animal's abdomen was extracted and the total numberof live/dead cells in each mouse was determined. In addition, some micewere used to assess a survival rate of animals in the experiment.

Goal of the study: in vivo assessment of potential antitumor activity of⁸⁵Rb_(e)-E2 in combination with DNA-damaging antineoplastic agentCisplatin against EAC tumor model.

Laboratory animals. 84 outbred female 10 to 12-week old mice weighing22-26 g were used in the experimental study. The animals were obtainedfrom the animal vivarium at R.E. Kavetsky IEPOR NASU. Before theexperiment, all the animals were healthy and demonstrated a normalbehavior pattern. The animals were maintained in plastic cages, undernatural light-dark cycles, fed standard food and allowed free access tofood and tap water.

Tumor model. Ascites strain of Ehrlich ascites carcinoma maintained onoutbred mice obtained from the cell and tissue line bank at R.E.Kavetsky IEPOR NASU was used in the experiment. To prepare the strainfor injection, tumor cells derived from ascitic fluid were placed innormal saline solution and cellularity of the suspension was assessed ina hemocytometer and adjusted to a concentration of 1·10⁶ cells/ml withsaline solution. The tumor cells were inoculated by injecting 250 mcl ofcell suspension from the tumor (0.25×10⁶ cells/mouse) into theperitoneal cavity of laboratory animals.

Grouping: The animals were divided into groups as follows (14 mice pergroup):

1. Group No. 1—control group, EAC+solvent (deuterium-depleted water);

2. Group No. 2—EAC+⁸⁵Rb_(e)-E2 injected IP at a dose of 4.5 mg/kg(EAC+CP(4.5));

3. Group No. 3—EAC+⁸⁵Rb_(e)-E2 injected IP at a dose of 750 μg/mouse+CPinjected IP at a dose of 4.5 mg/kg (EAC+⁸⁵Rb_(e)E2+CP(4.5));

4. Group No. 4—EAC+CP injected IP at a dose of 1.5 mg/kg (EAC+CP(1.5));

5. Group No. 5—EAC+⁸⁵Rb_(e)-E2 injected IP at a dose of 750 μg/mouse+CPinjected IP at a dose of 1.5 mg/kg (EAC+⁸⁵Rb_(e)E2+CP(1.5));

6. Group No. 6—EAC+⁸⁵Rb_(e)-E2 injected IP at a dose of 750 μg/mouse(EAC+⁸⁵Rb_(e) E2).

Injection Scheme of the Compounds Under Study

⁸⁵Rb_(e)-E2 was dissolved in deuterium-depleted water and Cisplatin wasdissolved in saline solution.

The drugs were administered IP every other day, using a microinjectionsyringe, at doses specified above as a series of 3 injections of eachcompound according to the following scheme: ⁸⁵Rb_(e)-E2 at a dose of 250μg/mouse weighing 20 g+CP at a dose of 1.5 mg/kg 24 hours later (forgroups (EAC+CP(4.5)) and (EAC+⁸⁵Rb_(e)-E2+CP(4.5)) or 0.5 μg/kg (forgroups (EAC+CP(1.5)) and (EAC+⁸⁵Rb_(e)-E2+CP(1.5)) in a volume of 0.25ml of saline solution (6 injections in total during 8 days).

The animals in the control group received IP injections ofdeuterium-depleted water in a volume of 0.3 ml/mouse.

Assessment of Tumor Growth

The tumor growth dynamics in the laboratory animals was observed bydaily control of the volume of ascitic fluid in the abdominal cavity ofmice starting from the tumor inoculation and for 31 days of theexperiment. The dynamics of ascites growth both in control andexperimental groups was assessed visually using a 10-point scale, where0 points were the absence of tumor growth and 10 points were the maximumincrease in the size of the abdomen of the mouse with ascites (one pointwas 0.6 cm of the mean diameter of the mouse abdomen filled with thefluid).

Furthermore, on the 15^(th) and 22^(nd) day after intraperitonealinjection of tumor cells, all ascitic fluid was removed from the abdomenin 2 or 3 animals from each group by washing their peritoneal cavitywith saline solution, and live/dead EAC cells were counted in each mouseusing the traditional vital dye trypan blue (HyClon, USA) and ahemocytometer.

The number of cells was determined according to the following formula:

X=((a)/80)×10⁶, where X is the number of cells in 1 ml and a is thenumber of cells counted in 5 large diagonal squares of thehemocytometer.

7 or 8 mice from each group were used to assess their survival in theexperiment. The total lifespan observation time was 31 days from themoment of inoculation of tumor cells.

Statistical data processing. To assess the significance levels ofdifferences in average values between groups Student's t-test andnon-parametric Mann-Whitney U test were applied. Calculations wereperformed using Medstatistic software package.

Results of the Study

Our previous in vitro studies on B16 melanoma cells have shown thatsuccessful in terms of cytotoxic effect on tumor cells is thecombination of ⁸⁵Rb_(e)-E2 and various antitumor drugs at the lowestpossible concentrations. It was shown that the most significant effectwas produced by a combined alternating action of ⁸⁵Rb_(e)-E2 andantitumor agents. Therefore, the in vivo antitumor activity of⁸⁵Rb_(e)-E2 was evaluated in the EAC tumor model both in a mono-regimeand in combination with cisplatin, an officially approved antitumordrug. In this experiment, the growth rate of ascites in the control andtherapeutic groups was assessed based on the size of abdomen of a mousewith ascites.

The results obtained showed that ⁸⁵Rb_(e)-E2, like cisplatin in amono-regime, suppressed the EAC development at the initial stages oftumor growth. Thus, ⁸⁵Rb_(e)-E2 has suppressed the growth of carcinomastatistically significantly, namely by an average of 18% by the 15^(th)day after the tumor cells were inoculated, and cisplatin at a dose of4.5 mg/kg—by an average of 51.3% throughout the observation period,compared with the control group. At the same time, a series ofconsecutive intraperitoneal injections of ⁸⁵Rb_(e)-E2 at a total dose of750 mg/mouse and cisplatin at a dose of 4.5 mg/kg resulted in a decreasein the size of experimental tumors by an average of 61% versus control.Such effect of suppression of tumor growth in this group was observedthroughout the experiment (Table 17).

Cisplatin in a mono-regime at a dose of 1.5 mg/kg exhibits astatistically significant suppression of tumor growth—by 32%—but only upto the 10^(th) day of the experiment. Furthermore, a statisticallysignificant suppression of the experimental tumor growth—by an averageof 28% versus control—was observed in the EAC+CP(1.5)+⁸⁵Rb_(e)-E2 group,and the effect persisted for 21 days after inoculation of tumor cells(Table 17).

TABLE 17 Dynamics of Ehrlich ascites carcinoma growth in the experiment(analysis of changes in the volumes of ascites in laboratory animals)(see FIG. 9) Day after injection of tumor cells Group of laboratory 9 1315 17 20 animals Size of mouse abdomen, points EAC control 5.6 ± 0.2   7± 0.2  8 ± 0.4 8.4 ± 0.5  9.5 ± 0.4  EAC + CP (4.5) 2.6 ± 0.2* 3.2 ±0.3* 3.1 ± 0.4* 3.4 ± 0.4* 4.8 ± 0.5* EAC + CP (4.5) +  3 ± 0.2*  3 ±0.3* 2.8 ± 0.3*  3 ± 0.4* 3.6 ± 0.5* ⁸⁵Rb_(e) − E2 EAC + CP (1.5) 3.8 ±0.8* 5.9 ± 1.1  6.9 ± 0.4  7.4 ± 0.6  8.1 ± 0.6  EAC + CP (1.5) + 3.4 ±02*  5.5 ± 0.3* 5.4 ± 0.4* 6.1 ± 0.4* 7.5 ± 0.6* ⁸⁵Rb_(e) − E2 EAC +⁸⁵Rb_(e) − E2 4.4 ± 0.2*  6 ± 0.3* 6.5 ± 0.3*  8 ± 0.6  9 ± 0.4 Dayafter injection of tumor cells Group of laboratory 22 24 27 29 31animals Size of mouse abdomen, points EAC control 9.7 ± 0.2 10 10 died —EAC + CP (4.5)  4.7 ± 0.7*  5.2 ± 0.9* 5.2 ± 0.9*  5.4 ± 0.9  6 ± 1.3EAC + CP (4.5) +  4.3 ± 0.6*   4 ± 0.6* 3.5 ± 0.7* 3.3 ± 1 3.3 ± 1 ⁸⁵Rb_(e) − E2 EAC + CP (1.5) 8.2 ± 0.8 8.6 ± 0.8 8.4 ± 1   8.4 ± 1 7.9 ±1.4 EAC + CP (1.5) + 8.2 ± 0.7 9.3 ± 0.6 9.4 ± 0.7  10 10 ⁸⁵Rb_(e) − E2EAC + ⁸⁵Rb_(e) − E2 9.5 ± 0.4 10 10 10 10 *p < 0.05 statisticallysignificant difference vs. control group

According to the results of the in vivo study, ⁸⁵Rb_(e)-E2 is able toenhance the antineoplastic activity of cisplatin against the EAC tumormodel. Thus, on the 27^(th) day of the experiment, when the size oftumors in the control group and in the group where the animals receivedinjections of ⁸⁵Rb_(e)-E2 alone reached 10 points, the mean tumor sizein the EAC+CP(4.5) group was 5.2±0.9, while in theEAC+CP(4.5)+⁸⁵Rb_(e)-E2 group it was 3.5±0.7 points. It should be notedthat in the therapeutic groups where mice were given a smaller dose ofcisplatin (1.5 mg/kg), the antitumor activity enhancing effect of⁸⁵Rb_(e)-E2 was also evident: statistically significant inhibition ofthe EAC growth, as compared with the control, was noted on day 21 afterinoculation of the tumor cells, whereas in the groups where the animalswere administered the substances at the same doses in a mono-regime thiseffect was preserved only up to day 9 (EAC+CP(1.5)) and day 15(EAC+⁸⁵Rb_(e)-E2) of the experiment (Table 17).

Data on the number of live and dead tumor cells in the ascitic fluid ofexperimental animals were also used to evaluate the in vivo antitumoractivity of the complex of ⁸⁵Rb_(e)-E2 compound and cisplatin, aDNA-damaging antitumor agent, against Ehrlich ascites carcinoma. Thefollowing results were obtained (Table 18).

TABLE 18 Total number of cells in the ascites on the 15^(th) and 22^(nd)day after inoculation of Ehrlich carcinoma cells (see FIGS. 10-12) Day15 Number of live Number of Group of laboratory cells/mouse, ×10⁶ deadcells/ Volume of animals (M ± m) mouse, % ascites, ml EAC control 1784 ±136 <4% 7.7 ± 1.5 EAC + CP(4.5)  33.5 ± 11* <1%  0.4 ± 0.1* EAC +CP(4.5) + 15.1 ± 8*  <1%  0.1 ± 0.1* ⁸⁵Rb_(e) − E2 EAC + CP(1.5) 1188 ±116 9.5 ± 2.8 5.2 ± 1.3 EAC + CP(1.5) + 1112 ± 147 13.1 ± 3   5.8 ± 2 ⁸⁵Rb_(e) − E2 EAC + ⁸⁵Rb_(e) − E2 1917 ± 408 4.3 ± 1.5 6.5 ± 2.9 Day 22Number of live Number of Group of laboratory cells/mouse, ×10⁶ deadcells/ Volume of animals (M ± m) mouse, % ascites, ml EAC control 1273 ±479 14.6 ± 10.1  16 ± 3.2 EAC + CP(4.5)  430 ± 250 <3%  2.8 ± 1.3 EAC +CP(4.5) +  252 ± 295  5 ± 3.8  2.8 ± 2.4 ⁸⁵Rb_(e) − E2 EAC + CP(1.5)1154 ± 429 11 ± 1  14.4 ± 8.7 EAC + CP(1.5) +  818 ± 468 34.7 ± 34.715.7 ± 2.6 ⁸⁵Rb_(e) − E2 EAC + ⁸⁵Rb_(e) − E2 2062 ± 438 15.4 ± 3.6  16.4± 4.8 *p < 0.05 statistically significant difference vs. control group

Analysis of the results showed that a notable and statisticallysignificant decrease in the number of tumor cells, compared with thecontrol, was observed only on the 15^(th) day of the experiment ingroups where mice were injected with cisplatin at a dose of 4.5 mg/kg.It should be noted that the combined administration of the drugs(CP(4.5)+⁸⁵Rb_(e)-E2) at the same dose enhances the effect: in theEAC+CP(4.5) and EAC+CP(4.5)+⁸⁵Rb_(e)-E2 groups, the number of live cellsin ascites was approximately 1/53 and 1/118 (p<0.05), respectively, ofthe number of live cells in the control (Table 18). In these groups, onthe 22^(nd) day of the experiment, the previously noted trendspersisted, but the decrease in the number of live tumor cells, incomparison with the control, was not statistically significant.

At the same time, in groups where the animals received cisplatin at adose of 1.5 mg/kg, both in a mono-regime and in combination with⁸⁵Rb_(e)-E2 on days 15 and 22 after the EAC cells were inoculated, therewere fewer tumor cells in the peritoneal cavity of mice compared withthe control group but the difference was statistically insignificant(Table 18).

Analysis of the number of mice in the experimental groups with andwithout experimental tumors supplements the data on antitumor activityof the experimental compound in mono-regime and in combination withcisplatin. The results presented in Table 19 show that of alltherapeutic schemes used in the experiment the most effective series wasa series of consecutive IP injections of ⁸⁵Rb_(e)-E2 at a total dose of750 μg/mouse (weighing 20 g) followed by the injection of cisplatin 24hour later at a total dose of 4.5 mg/kg, since only in this group therewere animals (16.7%) with 100% suppression of the EAC growth.

TABLE 19 Suppression of EAC tumor growth after it was exposed to thecombined action of cisplatin and the experimental compound containingstable isotope Rb⁸⁵ (and see FIGS. 13-16). Number of Number of Group ofanimals in the animals in the Animals laboratory group with groupwithout without animals tumor tumor tumor, % EAC control 14 0 0 EAC +CP(4.5) 14 0 0 EAC + CP(4.5) + 12 2 16.7 ⁸⁵Rb_(e) − E2 EAC + CP(1.5) 140 0 EAC + CP(1.5) + 14 0 0 ⁸⁵Rb_(e) − E2 EAC + ⁸⁵Rb_(e) − E2 14 0 0

Analysis of the survival rate of mice in the experiment showed that themost significant increase in the life span of animals was observed intherapeutic groups where mice received 4.5 mg/kg of cisplatin (FIG. 17).Thus, the last animal in the control group died on the 29^(th) day afterintroduction of carcinoma cells, while in the EAC+CP(4.5) andEAC+CP(4.5)+⁸⁵Rb_(e)-E2 groups, 75% and 62.5% of mice, respectively,remained alive. On day 31 of the experiment, 62.5% of mice survived inthese groups. At the same time, in groups where cisplatin wasadministered at a dose of 1.5 mg/kg in a mono-regime and in combinationwith ⁸⁵Rb_(e)-E2, an increase in the life span of mice was noted aswell. Thus, on day 29 of the experiment, in the EAC+CP(1.5) andEAC+CP(1.5)+⁸⁵Rb_(e)-E2 groups, there were 50% and 62.5% of live mice,respectively, and on day 31, there remained 37.5% of the survivors inboth groups. It should be noted that a series of IP injections of⁸⁵Rb_(e)-E2 alone did not have a significant effect on the survival rateof mice in the experiment.

Conclusion: It has been found that ⁸⁵Rb_(e)-E2 and cisplatin, whenadministered in a mono-regime, suppress the EAC development at theinitial stages of tumor growth. A statistically significant suppressionof experimental tumor growth—by an average of 28% with respect tocontrol—was observed in the EAC+CP(1.5)+⁸⁵Rb_(e)-E2 group for 21 daysafter the tumor cells were inoculated. According to the results of thein vivo study, ⁸⁵Rb_(e)-E2 is able to enhance the antineoplasticactivity of cisplatin against the EAC tumor model. Thus, on the 27^(th)day of the experiment, when the size of tumors in the control group andin the group where the animals received injections of ⁸⁵Rb_(e)-E2 alonereached 10 points, the mean tumor size in the EAC+CP(4.5) group was5.2±0.9, while in the EAC+CP(4.5)+⁸⁵Rb_(e)-E2 group it was 3.5±0.7points. The results also show that a notable and statisticallysignificant decrease in the number of tumor cells, compared with thecontrol, was observed on the 15^(th) day of the experiment in groupswhere mice were injected with cisplatin at a dose of 4.5 mg/kg, and thecombined administration of drugs (CP(4.5)+⁸⁵Rb_(e)-E2) at the same doseenhances the effect: in the EAC+CP(4.5) and EAC+CP(1.5)+⁸⁵Rb_(e)-E2groups, the number of live cells in ascites was 52 and 119 times smaller(p<0.05), respectively, than in the control.

Analysis of the survival rate of mice in the experiment showed that themost significant increase in the life span of animals was observed intherapeutic groups where mice received 4.5 mg/kg of cisplatin. Thus, thelast animal in the control group died on the 29^(th) day afterintroduction of carcinoma cells, while in the EAC+CP(4.5) andEAC+CP(4.5)+⁸⁵Rb_(e)-E2 groups, 75% and 62.5% of mice, respectively,remained alive. On day 31 of the experiment, 62.5% of mice survived inthese groups. In groups where cisplatin was administered at a dose of1.5 mg/kg in a mono-regime and in combination with ⁸⁵Rb_(e)-E2, anincrease in the life span of mice was noted as well. The data are shownin Table 20 and in FIG. 17.

TABLE 20 Experimental groups Day after [Percent (number of mice)] tumorcell EAC + EAC + CP(4, 5) + inoculation EAC control CP(4, 5) ⁸⁵Rb_(e) −E2 0 100% (7) 100% (8) 100% (8) 15 100% (7) 100% (8) 100% (8) 17 100%(7) 87.5% (7) 100% (8) 19 100% (7) 87.5% (7) 100% (8) 22 86% (6) 75% (6)100% (8) 24 71% (5) 75% (6) 75% (6) 25 57% (4) 75% (6) 75% (6) 26 57%(4) 75% (6) 75% (6) 27 43% (3) 75% (6) 75% (6) 28 14.3% (1) 75% (6) 75%(6) 29 0 75% (6) 62.5% (5) 31 62.5% (5) 62.5% (5) Experimental groupsDay after Percent (number of mice)] tumor cell EAC + EAC + CP(1.5) +EAC + inoculation CP(1.5) ⁸⁵Rb_(e) − E2 ⁸⁵Rb_(e) − E2 0 100% (8) 100%(8) 100% (7) 15 100% (8) 100% (8) 100% (7) 17 100% (8) 100% (8) 85.7%(6) 19 100% (8) 100% (8) 85.7% (6) 22 100% (8) 100% (8) 85.7% (6) 24100% (8) 87.5% (7) 85.7% (6) 25 100% (8) 87.5% (7) 71% (5) 26 100% (8)75% (6) 71% (5) 27 87.5% (7) 75% (6) 57% (4) 28 75% (6) 62.5% (5) 43%(3) 29 50% (4) 62.5% (5) 29% (2) 31 37.5% (3) 37.5% (3) 14% (1)

Example 6

Compound 7 was prepared as follows. The same procedure can be used toprepare the other compounds of Formulas 1 and 2, with use of ⁸⁵Rb_(e)Clor RbCl as appropriate and with starting materials and intermediatescomprising the appropriate substituents to obtain the desired product.An alternative synthesis is provided below for when R₉ is OCH₃.

Phase 1

10.8 g of o-phenylenediamine base were dissolved in 100 ml of isopropylalcohol and, while maintaining the mixture at room temperature, 200 mlof an alcohol solution of benzenesulfonyl chloride (17.65 g) were added.The resulting mixture was stirred up for 1 hour at room temperature and0.5 hours at 65-70° C. After cooling, the resulting light-coloredprecipitate was filtered by thorough washing with cold water to removethe residue of unreacted o-phenylenediamine. The precipitate from thefilter was boiled in 10% hydrochloric acid and filtered while hot toremove by-produced bis-dibenzenesulfonyl-o-phenylenediamine. Thefiltrate was clarified with activated carbon and, after cooling,N-benzenesulfonyl-o-phenylenediamine hydrochloride in the form of fineneedle crystals was filtered off (weight per dry matter 7.11 g. Yield25%).

Phase 2

N-benzenesulfonyl-o-phenylenediamine hydrochloride obtained in the firstphase (7.11 g) was suspended in 50 ml of toluene and 5.1 g ofo-nitrobenzoyl chloride and 5.32 g (7.1 ml) of triethylamine were added.The reaction mixture was boiled under reflux in an oil bath for 3 hoursand, after cooling, the precipitate formed was filtered off. Theresulting substance was recrystallized from isopropyl alcohol andpurified with activated carbon. The yield of dryN-benzenesulfonyl-N′-2-nitrobenzoyl-o-phenylenediamine was 6.45 g (65%).

Phase 3

Preparation of aqueous solution of rubidium salt ofN-benzenesulfonyl-N′-2-nitrobenzoyl-o-phenylenediamine with aconcentration of 0.001 g/ml with ⁸⁵Rb_(e): 0.073 g of KOH and 0.467 g ofN-benzenesulfonyl-N′-2-nitrobenzoyl-o-phenylenediamine were dissolved in90 ml of deionized water. The solution was stirred up while heating todissolve the precipitate completely. After being cooled to roomtemperature, the solution was filtered and 0.1417 g of ⁸⁵Rb_(e)Cl(⁸⁵Rb_(e) was 99% ⁸⁵Rb) was added, which quickly dissolved. The reactionmixture was stirred up at room temperature for 30 minutes and filteredagain. The filtrate was made up to 100 ml with deionized water and usedfor further studies.

The following methods, among others, can be used to identify the newlysynthesized compounds: measurement of the PMR spectra on a Varian VXR200 spectrometer with respect to TMS in DMSO-d₆; measurement of IRspectra (4000-600 cm⁻¹) on a Bruker ALPHA FT-IR spectrometer using theATR accessory; TLC on Silicagel 60 F₂₅₄ plates (eluent: chloroform);determination of mass spectra using a Kratos MS 890 mass spectrometer,with direct injection of the sample into the ion source at an ionizationchamber temperature of 180-250° C. and ionizing electron energy of 70eV.

When R₉ is OCH₃, the following alternative synthesis may be used. In thelast step, ⁸⁵Rb_(e)Cl can be used instead of RbCl in order to prepare an⁸⁵Rb-enriched product.

The invention claimed is:
 1. A compound of the following formula:

wherein each of R₁ through R₁₄ is independently selected from H, OH, F,Cl, Br, I, C₁-C₆ alkyl, C₁-C₆ alkoxy, and NO₂ and the ⁸⁵Rb_(e) is atleast 75% ⁸⁵Rb.
 2. The compound of claim 1, wherein the ⁸⁵Rb_(e) is atleast 90% ⁸⁵Rb.
 3. The compound of claim 1 wherein R₁, R₂, R₄-R₆, R₈,R₁₀, R₁₁, and R₁₃ are all H.
 4. The compound of claim 3, wherein the⁸⁵Rb_(e) is at least 90% ⁸⁵Rb.
 5. The compound of claim 3 wherein R₃ isselected from H, CH₃, OCH₃, and NO₂, R₇ and R₉ are each independentlyselected from H and OCH₃, and R₁₂ and R₁₄ are each independentlyselected from H, Br, I, and NO₂.
 6. The compound of claim 5, wherein the⁸⁵Rb_(e) is at least 90% ⁸⁵Rb.
 7. The compound of claim 3, wherein a) R₃is CH₃ and R₇, R₉, R₁₂, and R₁₄ are all H, b) R₃, R₇, R₉, R₁₂, and R₁₄are all H, c) R₃ is CH₃, R₁₄ is Cl, and R₇, R₉, and R₁₂ are all H, d) R₃is CH₃, R₁₄ is OH and R₇, R₉, and R₁₂ are all H, e) R₁₄ is OH and R₃,R₇, R₉, and R₁₂ are all H, f) R₃ is OH and R₇, R₉, R₁₂, and R₁₄ are allH, g) R₁₄ is NO₂ and R₃, R₇, R₉, and R₁₂ are all H, h) R₁₂ is Br, R₁₄ isNO₂ and R₃, R₇, and R₉ are all H, i) R₃ and R₉ are both OCH₃, R₁₂ is Br,R₁₄ is NO₂ and R₇ is H, or j) R₃ and R₉ are both OCH₃, R₁₄ is NO₂ and R₇and R₁₂ are both H.
 8. The compound of claim 7, wherein the ⁸⁵Rb_(e) isat least 90% ⁸⁵Rb.
 9. A composition comprising the compound of claim 1and an excipient, wherein the compound is present in an amountequivalent to between 40 mg ⁸⁵Rb_(e) and 2400 mg ⁸⁵Rb_(e).
 10. Thecomposition of claim 9 wherein, in the compound of claim 1, the ⁸⁵Rb_(e)is at least 90% ⁸⁵Rb.
 11. The composition of claim 10 wherein, in thecompound of claim 1, R₁, R₂, R₄-R₆, R₈, R₁₀, R₁₁, and R₁₃ are all H. 12.The composition of claim 11 wherein, in the compound of claim 1, R₃ isselected from H, CH₃, OCH₃, and NO₂, R₇ and R₉ are each independentlyselected from H and OCH₃, and R₁₂ and R₁₄ are each independentlyselected from H, Br, I, and NO₂.
 13. The composition of claim 11,wherein, in the compound of claim 1, a) R₃ is CH₃ and R₇, R₉, R₁₂, andR₁₄ are all H, b) R₃, R₇, R₉, R₁₂, and R₁₄ are all H, c) R₃ is CH₃, R₁₄is Cl, and R₇, R₉, and R₁₂ are all H, d) R₃ is CH₃, R₁₄ is OH and R₇,R₉, and R₁₂ are all H, e) R₁₄ is OH and R₃, R₇, R₉, and R₁₂ are all H,f) R₃ is OH and R₇, R₉, R₁₂, and R₁₄ are all H, g) R₁₄ is NO₂ and R₃,R₇, R₉, and R₁₂ are all H, h) R₁₂ is Br, R₁₄ is NO₂ and R₃, R₇, and R₉are all H, i) R₃ and R₉ are both OCH₃, R₁₂ is Br, R₁₄ is NO₂ and R₇ isH, or j) R₃ and R₉ are both OCH₃, R₁₄ is NO₂ and R₇ and R₁₂ are both H.